Abstract Blood oxygen level dependent (BOLD) MRI contrast revolutionized neurology and neuroscience by offering a mechanism to visualize functional activation in the living brain. Diffusion-weighted functional MRI (DfMRI) is a recently proposed functional mechanism which promises to improve spatial and temporal resolution; however, concerns remain as to the true biophysical mechanism responsible for signal changes in DfMRI. Until now, the inability to image living samples at sufficient MR resolutions has prevented direct observation of the tissue elements responsible for signal changes in DfMRI of mammals. By employing world-class dedicated hardware?ultra-high field imaging systems and micro coil technology?in conjunction with a one-of-a-kind oxygenation and microperfusion system purposed-designed to interface with this hardware, we have created a unique opportunity to investigate the DfMRI contrast mechanism at higher spatial resolutions than have ever before been possible. In addition, the acute slice preparation is a devascularized tissue model that precludes the most likely confound?residual vascular effects ?from contributing to our diffusion data. The long term goal of the current study is thus to identify the true biophysical underpinnings of DfMRI as a means to assess its future clinical utility. The primary objective of this proposal is to identify the laminar and cellular components responsible for generating signal changes in DfMRI. Our hypothesis is that the diffusion-weighted MR signal of nervous tissues will differ significantly depending on whether the tissue is active (neuronal firing) or inactive (resting state). Our rationale is that the newfound ability to observe which microstructural features of neural tissue undergo signal changes between periods of firing and rest will offer critical insight into the physiological basis of this still controversial imaging technique. The hypothesis will be tested in the following two specific aims: 1) Compare diffusion signal measurements in individual lamina of acute hippocampal slices during periods of activation and rest; and 2) Investigate diffusion signal changes at subcellular resolution in ?-motor neurons during periods of activation and rest. In Aim 1, high temporal (2min 8 sec), lower spatial (47?m in- plane) resolution diffusion-weighted images will be collected in the CA1 region of the acute hippocampal slice during periods of rest (untreated aCSF) and tonic activation (100?M kainate). For Aim 2, higher spatial (6.25 ?m isotropic) but lower temporal (3.5 h) resolution imaging protocols will be employed in acute slices taken from the cervical enlargement of the spinal cord. Regions of interest (ROIs) will be identified in each image according to the microstructural elements visualized (strata orients, pyramidale, and radiatum in hippocampus and perikarya, neurites, and neuropil in the spinal cord) and compared before and after exposure to kainate. Data will reveal which microstructural elements are responsible for generating signal changes in DfMRI. The proposed research is significant in that it could potentially validate a powerful, complimentary functional imaging mechanism that is noninvasive and immediately applicable in the clinic.